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Estuarine Pettaquamscutt River is a unique habitat 10 km in length with physical and biogeochemical characteristics analogous to a miniature fjord. Its meromictic upper basins are characterized by a permanent oxic–anoxic vertical gradient in which a well-oxygenated upper layer overlies a deeper, anoxic reservoir, with persistent blooms of phototrophic anoxygenic bacteria (Chromatium, Chlorobium) at the oxic–anoxic transition layer. A diverse assemblage of nanoplanktonic, centric diatoms (Cyclotella caspia, Thalassiosira pseudonana, cf. Cyclotella cryptica) dominated the seasonal phytoplankton cycle in the aerobic layer, similar to comparable meromictic habitats elsewhere. This assemblage of nano-centric diatoms appears to be trait-separated from other species clusters and is potentially useful as a functional group flora with ecophysiology diagnostic of marine estuarine rivers and meromictic habitat niche structure. The most conspicuous phytoplankton feature, however, was the year-round occurrence of the photoautotrophic euglenid Euglenaformis (Euglena) proxima, restricted in its upper water column distribution to the O2–H2S boundary layer where it formed a consortium with the photosynthetic green and purple sulfur bacteria community. The association of E. proxima with meromixis, H2S, and phototrophic anoxygenic bacteria is similar to that reported previously in a brackish Norwegian oyster poll and a brackish loch in Scotland.

[Image: see text] Spiroimines are a class of compounds produced by marine dinoflagellates with a wide range of toxicity and therapeutic potential. The smallest of the cyclic imines, portimine, is far less toxic than other known members in several animal models. Portimine has also been shown to induce apoptosis and reduce the growth of a variety of cancer cell lines at low nanomolar concentrations. In an effort to discover new spiroimines, the current study undertook a metabolomic analysis of cultures of cyclic imine-producing dinoflagellates, and a new analog of portimine was discovered in which the five-membered cyclic ether is open. Further scrutiny with human oral cavity squamous cell carcinoma (OCSCC) cell lines revealed that the open ring congener was less potent than portimine A but could still lead to the accumulation of apoptotic gene transcripts, fragment genomic DNA, and reduce cancer cell proliferation in the range of 100–200 nM.

Heterosigma akashiwo is one of the smallest raphidophytes and is known for its ability to form dense, fish-killing blooms. In many parts of the world, H. akashiwo is a permanent resident of the algal community, and blooms have caused extensive financial losses for the aquaculture industry. Different aspects of its biology have been the focus of numerous investigations over the last two decades but, perhaps because of its small size and fragile nature, few morphological studies have been carried out. We conducted phylogenetic and morphological studies of 24 strains of Heterosigma from the coastal waters of Europe, North America, Australia and New Zealand. Complete 18S, ITS region and the D1–D2 region of 28S rDNA were sequenced and compared with all available raphidophyte sequences, revealing a novel species of Heterosigma, Heterosigma minor sp. nov. All strains of H. akashiwo were genetically very similar across the rDNA region (> 99.8% similarity) and morphologically very similar in light microsc...

Harmful bloom-forming algae include some of the most prolific microbial producers of extracellular reactive oxygen species (ROS). However, the taxonomic diversity of ROS production, the underlying physiological mechanisms and ecophysiological roles of ROS cycling are not completely characterized among phytoplankton taxa that form harmful algal blooms (HABs). This study examines the extracellular production of the ROS superoxide and hydrogen peroxide by five marine HAB species: Chattonella marina, Heterosigma akashiwo, Karenia brevis, Pseudo-nitzschia sp. and Aureococcus anophagefferens. All species produced extracellular superoxide and hydrogen peroxide. Rates of ROS production per cell spanned several orders of magnitude and varied inversely with cell density, suggesting a potential signaling role for extracellular ROS. ROS production was also detected in the spent media of all cultures except K. brevis, indicating the presence of cell-free ROS-generating constituents, such as enzymes or metabolites, which could be further investigated as molecular targets for tracking ROS production in laboratory and field settings. Finally, ratios of superoxide to hydrogen peroxide production could not be accounted for by superoxide dismutation alone, except in the case of K. brevis, indicating a diversity of ROS production and degradation pathways that may ultimately help illuminate the functions of HAB-derived ROS.

Coolia santacroce is a newly described epibenthic dinoflagellate species collected from the U.S. Virgin Islands. The original description indicates this species is unique from others in the Coolia monotis complex due to the relative size of the apical pore complex, broad range of pore sizes, and ribosomal DNA. The original description was done based on the isolation and cultivation of one isolate of the organism. In this study, we report three more isolates of Coolia santacroce collected from the Bahamas. Morphological observations were made using scanning electron microscopy that do not correspond to those from the original description, indicating the variability of the morphological features. However, analysis of the D1 / D2 regions of the large subunit rDNA places the three strains in a strongly supported monophyletic clade with the type specimen.

The dinoflagellate genus Coolia Meunier is an important epi-benthic organism that is commonly found in association with other dinoflagellates known to cause ciguatera. Two closely related taxa, Coolia monotis and Coolia malayensis , make up the C . monotis species complex. In this study we introduce two new toxic species that should be included in that complex, Coolia palmyrensis Karafas, Tomas, York sp. nov. and Coolia santacroce Karafas, Tomas, York sp. nov., collected from the Palmyra Atoll in the Pacific Ocean and Saint Croix, US Virgin Islands, respectively. These two species can be distinguished morphologically by size, pore shape, pore density, and the relative size of the apical pore complex. The ITS1/5.8S/ITS2 and the D1/D2 regions of the LSU rDNA were used to provide molecular support of morphological observations using maximum likelihood and Bayesian analyses. Furthermore, C . palmyrensis and C . santacroce both showed cytotoxic effects on human derived cells in vitro .

The vernal occurrence of toxic dinoflagellates in the Alexandrium tamarense/Alexandrium fundyense species complex in an enclosed embayment of Narragansett Bay (Wickford Cove, Rhode Island) was documented during 2005 and 2009–2012. This is the first report of regular appearance of the Alexandrium fundyense/Alexandrium tamarense species complex in Narragansett Bay. Thecal plate analysis of clonal isolates using SEM revealed cells morphologically consistent with both Alexandrium tamarense Lebour (Balech) and Alexandrium fundyense Balech. Additionally, molecular analyses confirmed that the partial sequences for 18S through the D1–D2 region of 28S were consistent with the identity of the two Alexandrium species. Toxin analyses revealed the presence of a suite of toxins (C1/2, B1 (GTX-5), STX, GTX-2/3. Neo, and GTX-1/4) in both Alexandrium tamarense (6.31 fmol cell −1 STX equiv.) and Alexandrium fundyense (9.56 fmol cell −1 STX equiv.) isolated from Wickford Cove; the toxicity of a Narragansett Bay Alexandrium peruvianum isolate (1.79 fmol cell −1 STX equiv.) was also determined. Combined Alexandrium tamarense / Alexandrium fundyense abundance in Wickford Cove reached a peak abundance of 1280 cells L −1 (May of 2010), with the combined abundance routinely exceeding levels leading to shellfishing closures in other systems. The toxic Alexandrium tamarense / Alexandrium fundyense species complex appears to be a regular component of the lower Narragansett Bay phytoplankton community, either newly emergent or previously overlooked by extant monitoring programs.

Alexandrium ostenfeldii (Paulsen) Balech and Tangen and A. peruvianum (Balech and B.R. Mendiola) Balech and Tangen are morphologically closely related dinoflagellates known to produce potent neurotoxins. Together with Gonyaulax dimorpha Biecheler, they constitute the A. ostenfeldii species complex. Due to the subtle differences in the morphological characters used to differentiate these species, unambiguous species identification has proven problematic. To better understand the species boundaries within the A. ostenfeldii complex we compared rDNA data, morphometric characters and toxin profiles of multiple cultured isolates from different geographic regions. Phylogenetic analysis of rDNA sequences from cultures characterized as A. ostenfeldii or A. peruvianum formed a monophyletic clade consisting of six distinct groups. Each group examined contained strains morphologically identified as either A. ostenfeldii or A. peruvianum. Though key morphological characters were generally found to be highly variable and not consistently distributed, selected plate features and toxin profiles differed significantly among phylogenetic clusters. Additional sequence analyses revealed a lack of compensatory base changes in ITS2 rRNA structure, low to intermediate ITS/5.8S uncorrected genetic distances, and evidence of reticulation. Together these data (criteria currently used for species delineation in dinoflagellates) imply that the A. ostenfeldii complex should be regarded a single genetically structured species until more material and alternative criteria for species delimitation are available. Consequently, we propose that A. peruvianum is a heterotypic synonym of A. ostenfeldii and this taxon name should be discontinued.

This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ~7–40% from log phase growth to late log – early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20–31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60–90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced by Gambierdiscus species. While the results from inhibition studies require validation by LC–MS analysis, the available data strongly suggest differences in hemolytic activity observed in this study reflect maitotoxicity.

Harmful blooms formed by species of the dinoflagellate Cochlodinium have caused massive fish kills and substantial economic losses in the Pacific Ocean. Recently, prominent blooms of Cochlodinium have occurred in central and southern California (2004-2008), and Cochlodinium cells are now routinely observed in microscopical analysis of algal assemblages from Californian coastal waters. The first documented economic loss due to a Cochlodinium bloom in California occurred in Monterey Bay and resulted in the mortality of commercially farmed abalone. Increasing occurrences of Cochlodinium blooms, the fact that these cells preserve poorly using standard techniques, and the difficulty of identifying preserved specimens using morphological criteria make Cochlodinium species prime candidates for the development of a quantitative real-time polymerase chain reaction (qPCR) approach. The 18S rDNA gene sequenced from Cochlodinium cells obtained from California coastal waters, as well as GenBank sequences of Cochlodinium, were used to design and test a Molecular Beacon(®) approach. The qPCR method developed in this study is species specific, sensitive for the detection of C. fulvescens that has given rise to the recent blooms in the eastern Pacific Ocean, and spans a dynamic abundance range of seven orders of magnitude. Initial application of the method to archived field samples collected during blooms in Monterey Bay revealed no statistically significant correlations between gene copy number and environmental parameters. However, the onset of Cochlodinium blooms in central California was consistent with previously reported findings of correlations to decreased surface temperature and increased inputs of nitrogenous nutrients.

Defined experimental regimes were used to determine the effects of nutrient limitation on the toxicity of Alexandrium peruvianum in batch culture. Subsamples for cell counts and spiroimine analysis at six day intervals were used to investigate the concentrations and composition of these compounds throughout growth. An erythrocyte lysis assay for hemolytic activity was performed on cell pellets and supernatants also collected every six days over the entire growth period from all treatments. From the data, growth rates, cellular spiroimine quotas and effective concentration-fifty (EC50s) for cellular and supernatant associated hemolytic activity were calculated. Phosphate limitation was identified as a key regulator of toxicity in this species, yielding maximum values of 54.1 pg cell−1 for 13-desmethyl spirolide C, 96.4 pg cell−1 for 12-methylgymnodimine and a potent hemolytic EC50 value of 7.1 × 103 cells. The concentrations of spiroimines detected in A. peruvianum among various treatments, in addition to a unique profile of paralytic shellfish poisoning toxins, is unique in the body of microalgal literature. Because of the multiple toxin arsenal produced by this organism, the evaluation of a single toxin clearly would have underestimated the potential virulence and significance of this clone. This study provides the first evidence that growth and toxin production of A. peruvianum are influenced by altered nutrient ratios.

Phytoplankton monitoring in Wickford Cove, Rhode Island, US (41°34′10.13″N, 71°26′45.76″W), located in Narragansett Bay, detected an unusual species of Alexandrium in the spring of 2009. Thecal plate analysis using brightfield and SEM microscopy revealed a plate morphology consistent with that of Alexandrium peruvianum (Balech and de Mendiola) Balech and Tangen. Molecular analyses indicated that the sequences of the SSU, ITS1, 5.8S, ITS2 and LSU through the D region of the 18S gene were similar to those of A. peruvianum from North Carolina. Toxin analyses of cells brought into culture revealed saxitoxins, gymnodimine and fast-acting spiroimines were present in the cultured clone. Saxitoxins detected included GTX 2, GTX3, B1, STX, C1 and C2. Also present in the Wickford cove isolates of A. peruvianum were 12-methyl gymnodimine and 13-desmethyl spirolide C. A. peruvianum was detected at four sites in lower Narragansett Bay: at two sites in Wickford and two sites in Jamestown, RI. A. peruvianum was observed in the spring of 2009, 2010, 2011 and 2012 at maximum abundance levels ranging from tens of cells per liter to 14,000 cells L −1 . The discovery of A. peruvianum in Rhode Island coastal waters, with its potential threat to public health, is notable as it appears to be an emergent bloom species globally. The presence of A. peruvianum in Narragansett Bay is the third confirmed observation of this species on the Atlantic coast of North America. Monitoring efforts in the southern New England region should incorporate measures to detect the presence of A. peruvianum toxins.

Routine sampling of the water quality stations in the New River Estuary (Jacksonville, North Carolina, USA) during November 2004 revealed the presence of a previously unidentified dinoflagellate. Preliminary observations of its morphology suggested it to be consistent with that of Alexandrium peruvianum (Balech et Mendiola) Balech et Tangen. Observations using brightfield, epifluorescence and scanning electron microscopy confirmed the diagnostic thecal plates to be those of A. peruvanium . Clonal cultures established from cells isolated from the New River Estuary samples were also used for further studies of morphology and for the presence of toxins. Thecal morphology was consistent with that described by Balech clearly separating it from the sister species Alexandrium ostenfeldii . Three classes of toxins were detected from these cultures. An erythrocyte lysis assay (ELA) was used to confirm the presence of hemolytic toxins in A. peruvianum cultures. A cellular EC 50 for lysis was 1.418 × 10 4 cells, well within the range the maximal cells densities found in the New River and more potent when compared on a cellular basis with Prymnesium parvum . Another toxin class detected in A. peruvianum cultures was the fast acting 13-desmethy C and D spirolides also produced by the sister species A. ostenfeldii . The last toxin type detected in the A. peruvianum cultures was the paralytic shellfish toxins, GTX 2, 3, B1, STX and C1,2. These findings expand the geographic range of occurrence for A. peruvianum in the U.S. to be much greater than previously considered. The morphological characters agreed with previously reported molecular data in separating A. peruvianum from A. ostenfeldii . It is also the first confirmed report that this species produces PSP toxins, spirolides and naturally occurring hemolytic substances. In light of these findings additional attention is needed for the detection of Alexandrium species in all coastal waters of the U.S. This added effort will enhance the evaluation of the relative impacts of the species to shellfish safety and bloom surveillance.

Harmful blooms formed by species of the dinoflagellate Cochlodinium have caused massive fish kills and substantial economic losses in the Pacific Ocean. Recently, prominent blooms of Cochlodinium have occurred in central and southern California (2004-2008), and Cochlodinium cells are now routinely observed in microscopical analysis of algal assemblages from Californian coastal waters. The first documented economic loss due to a Cochlodinium bloom in California occurred in Monterey Bay and resulted in the mortality of commercially farmed abalone. Increasing occurrences of Cochlodinium blooms, the fact that these cells preserve poorly using standard techniques, and the difficulty of identifying preserved specimens using morphological criteria make Cochlodinium species prime candidates for the development of a quantitative real-time polymerase chain reaction (qPCR) approach. The 18S rDNA gene sequenced from Cochlodinium cells obtained from California coastal waters, as well as GenBank sequences of Cochlodinium, were used to design and test a Molecular Beacon(®) approach. The qPCR method developed in this study is species specific, sensitive for the detection of C. fulvescens that has given rise to the recent blooms in the eastern Pacific Ocean, and spans a dynamic abundance range of seven orders of magnitude. Initial application of the method to archived field samples collected during blooms in Monterey Bay revealed no statistically significant correlations between gene copy number and environmental parameters. However, the onset of Cochlodinium blooms in central California was consistent with previously reported findings of correlations to decreased surface temperature and increased inputs of nitrogenous nutrients.

Due to slow rates of molecular evolution, DNA sequences used to identify and build phylogenies of algal species involved in harmful algal blooms (HABs) are generally invariant at the intraspecific level. This means that it is unknown whether HAB events result from the growth of a single clone, a few dominant clones, or multiple clones. This is true despite the fact that several physiological and demographic traits, as well as toxicity, are known to vary across clones. We generated AFLP fingerprints from a set of 6 clonal isolates, taken from a bloom of Prymnesium parvum at a striped bass mariculture facility. This new haptophyte bloom was recently implicated in fish kills at several sites in the United States. The AFLP fragments were highly reproducible and showed that all isolates were distinguishable due to abundant AFLPs unique to single isolates. These results demonstrate that blooms can be genetically diverse outbreaks and indicate that AFLP can be a powerful molecular tool for characterizing and monitoring this diversity.

The novel gymnodimine congener 12-methylgymnodimine was isolated from the dinoflagellate Alexandrium peruvianum from the New River in North Carolina. In addition, the presence of 13-desmethylspirolide C was confirmed by NMR characterization. This marks the first time these two classes of cyclic imines have been found in the same organism, providing further evidence for a direct genetic relationship between the biosynthetic pathways for gymnodimines and spirolides suggested by comparison of their structures.

Karenia brevis forms extensive, annual blooms in the Gulf of Mexico releasing potent neurotoxins (brevetoxins) having significant impacts on human health, mortalities of marine mammals, birds and fish. This study examines toxin composition and growth of three clones of K. brevis grown at different nutrient conditions, salinities and temperatures. The three clones studied were K. brevis Wilson, SP3 N-tox and SP3 S-tox clones. Brevetoxins (PbTx-1, -2, -3, -6 and -9) and brevenal were examined using LC–MS/MS. Dialysis studies clearly indicated that PbTx-1, -2 and brevenal were found within intact cells while the other toxins were released in the surrounding medium. Nutrient limitation did not show consistent effects on toxin profiles (brevenal and PbTx-1, -2, -3, -6 and -9) but toxin profiles were significantly related to clone type. The Wilson clone produced a significantly greater amount of brevenal than either SP3 clone. The SP3 clones each responded differently to nutrient limitation in toxin fractions produced. Growth at differing salinities and temperatures confirmed previous reports of K. brevis preference of higher salinities and elevated temperatures but was variable for toxins. These results support the notion that clonal differences in toxin production and profile appears to be less dependent on environmental variables but more related to inherent genetic variations resulting in the differences in clonal responses observed .

Blooms of the toxic dinoflagellate, Karenia brevis, occur annually along the Gulf coast of Florida. Other species, like Karenia selliformis, are at times found in association. Hemolytic activity, the ability to lyse red blood cells, of two K. brevis clones (SP3 non-toxic (N-tox) and SP3 super toxic (S-tox)) from the Gulf of Mexico and a single clone of K. selliformis from New Zealand was investigated throughout a growth cycle. Activity is reported as effective concentration (EC50) values, the quantitative measure of hemolysis of human erythrocytes expressed as cell numbers. Both cells and media of K. selliformis cultures consistently produced potent levels of hemolysis (maximum EC50 = 4.88 × 103 cells) from inoculation until the population declined 35 days later. For SP3 N-tox and S-tox, no hemolytic activity was detectable until day 26 of sampling. The media of both SP3 N-tox and SP3 S-tox cultures consistently contained non-detectable or low levels of hemolysis compared to K. selliformis. Maximum EC50s for the SP3 clones were 1.80 × 106 and 1.97 × 106 cells, respectively. The experimental EC50 values observed represent ecologically relevant cell densities for K. selliformis, but not for the K. brevis clones. In addition, the hemolytic activity of gymnodimine A and various PbTx derivatives was examined in this study. Our findings indicate that the hemolytic capability of these dinoflagellates, especially K. selliformis, represents an additional component of toxicity aside from their already recognized toxins and that this activity may play a larger role than was previously considered. The purpose of this study was to extend the knowledge of the biology and toxicology of species within the genus Karenia.

Strong haemolytic activity was observed for extracts of 15 Fibrocapsa japonica strains collected from different global regions. The EC(50) values ranged between 0.4 x 10(4) and 1.9 x 10(4) F.japonica cells ml(-1).The relationship between the haemolytic activity observed in the cell extracts and the amount and nature of polyunsaturated fatty acids (PUFAs)was investigated. Between 7% and 89% of the haemolytic activity could be attributed to the amount and toxicity of the PUFAs C18:4n3 (OTA), C20:5n3 (EPA), and C20:4n6 (AA). However, the higher the haemolytic activity of a strain the more it appeared to be caused by haemolytic compounds other than the PUFA's. The EC(50) of the extracts with the highest haemolytic activity strongly depended on light intensity and was in line with observations made on Chattonella marina.In the cell extracts of the strains studied brevetoxins were always below detection limits. For strains where 20-35% of the haemolytic activity was not caused by PUFAs, the concentration of these compounds was close to LC(50) values reported for fish. Overall the haemolytic activity of the F. japonica strains was in the range of known harmful microalgae. Therefore, PUFAs in combination with potent light dependent haemolytic compounds could be a major cause of ichthyotoxicity observed during F. japonica blooms. (C) 2009 Elsevier B.V. All rights reserved.

Sterol and fatty acid compositions were determined for three harmful algal species previously classified in the genus Chattonella (Raphidophyceae): the new genus Chloromorum toxicum (ex Chattonella cf. verruculosa), Verrucophora farcimen (Dictyochophyceae), previously Chattonella aff. verruculosa, and Verrucophora verruculosa (=Pseudochattonella verruculosa) previously Chattonella verruculosa. The major fatty acids of C. toxicum were 14:0, 16:0, 18:1n−9, 18:4n−3 and 20:5n−3, and those of the Verrucophora strains were. 14:0, 16:0, 18:0, 18:4n−3, 18:5n−3 and 22:6n−3. C. toxicum contained the 24β-ethyl sterols, poriferasterol and clionasterol, as its major sterols. For comparison, the stereochemistry of the 24-ethyl sterols of two raphidophytes, Chattonella marina and Heterosigma akashiwo, was determined to be 24α and 24β, respectively. Both Verrucophora strains contained the 27-nor sterol occelasterol as the only detected sterol. This was the first time occelasterol has been found in algae.

Successive blooms of the dinoflagellate Cochlodinium polykrikoides occurred in Pettaquamscutt Cove, RI, persisting from September through December 1980 and again from April through October 1981. Cell densities varied from 20 g C m−3. Primary production by the dinoflagellate-dominated community during the bloom varied between 4.3 and 0.07 g C m−3 d−1. Percent carbon turnover calculated from primary production values and ATP-carbon varied from 6 to 129% d−1. The dinoflagellates dominated the entire summer period; other flagellates and diatoms were present in lesser amounts. A combination of low washout rate due to the cove dynamics, active growth, and life cycles involving cysts allowed C. polykrikoides to maintain recurrent bloom populations in this area.

Gonyostmum semen is a freshwater raphidophyte that has increased in occurrence and abundance in several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north-eastern Europe, and it is frequently referred to as invasive. To better understand the species history, we have explored the phylogeography of G. semen using strains from northern Europe, United States, and Japan. Three regions of the ribosomal RNA gene (small subunit [SSU], internal transcribed spacer [ITS] and large subunit [LSU]) and one mitochondrial DNA marker (cox1) were analyzed. The SSU and partial LSU sequences were identical in all strains, confirming that they belong to the same species. The ITS region differentiated the American from the other strains, but showed high intra-strain variability. In contrast, the mitochondrial marker cox1 showed distinct differences between the European, American, and Japanese strains. Interestingly, only one cox1 haplotype was detected in European strains. The overall low diversity and weak geographic structure within northern European strains supported the hypothesis of a recent invasion of new lakes by G. semen. Our data also show that the invasive northern European lineage is genetically distinct from the lineages from the other continents. Finally, we concluded that the mitochondrial cox1 was the most useful marker in determining large-scale biogeographic patterns in this species.

Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real-time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small-subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species.

Ten fish mortality events, involving primarily Atlantic menhaden, occurred from early July through September 2000 in several bays and creeks in Delaware, USA. Two events involved large mortalities estimated at 1–2.5 million fish in Bald Eagle Creek, Rehoboth Bay. Samples from Indian Inlet (Bethany Beach), open to the Atlantic, as well as from an enclosed area of massive fish kills at nearby Bald Eagle Creek and Torque Canal were collected and sent to our laboratory for analysis. Microscopic examination of samples from the fish kill site revealed the presence of a single-cell Raphidophyte alga Chattonella cf. verruculosa at a maximum density of 1.04 × 107 cells/L. Naturally occurring brevetoxins were also detected in the bloom samples. Besides the Chattonella species, no other known brevetoxin-producing phytoplankton were present. Chromatographic, immunochemical, and spectroscopic analyses confirmed the presence of brevetoxin PbTx-2, and PbTx-3 and -9 were confirmed by chromatographic and immunochemical analyses. This is the first confirmed report in the United States of brevetoxins associated with an indigenous bloom in temperate Atlantic estuarine waters and of C. cf. verruculosa as a resident toxic organism implicated in fish kills in this area. The bloom of Chattonella continued throughout September and eventually declined in October. By the end of October C. cf. verruculosa was no longer seen, nor was toxin measurable in the surface waters. The results affirm that to avoid deleterious impacts on human and ecosystem health, increased monitoring is needed for brevetoxins and organism(s) producing them, even in areas previously thought to be unaffected. Key words: brevetoxins, Chattonella cf. verruculosa, Delaware, fish kills, harmful agal blooms. Environ Health Perspect 110:465–470 (2002). [Online 1 April 2002] http://ehpnet1.niehs.nih.gov/docs/2002/110p465-470bourdelais/abstract.html

We developed a competitive enzyme-linked immunosorbent assay (ELISA) to analyze brevetoxins, using goat anti-brevetoxin antibodies obtained after immunization with keyhole limpet hemocyanin-brevetoxin conjugates, in combination with a three-step signal amplification process. The procedure, which used secondary biotinylated antibodies, streptavidine-horseradish peroxidase conjugate, and chromogenic enzyme substrate, was useful in reducing nonspecific background signals commonly observed with complex matrices. This competitive ELISA detected brevetoxins in seawater, shellfish extract and homogenate, and mammalian body fluid such as urine and serum without pretreatment, dilution, or purification. We investigated the application of this technique for shellfish monitoring by spiking shellfish meat with brevetoxins and by analyzing oysters from two commercial shellfish beds in Florida that were exposed to a bloom of Karenia brevis (formerly Gymnodinium breve). We performed brevetoxin analysis of shellfish extracts and homogenates by ELISA and compared it with the mouse bioassay and receptor binding assay. The detection limit for brevetoxins in spiked oysters was 2.5 microg/100 g shellfish meat. This assay appears to be a useful tool for neurotoxic shellfish poisoning monitoring in shellfish and seawater, and for mammalian exposure diagnostics, and significantly reduces the time required for analyses.

Polyether marine toxins are responsible for the seafood intoxication phenomena known as neurotoxic shellfish poisoning (due to brevetoxins), ciguatera (due to ciguatoxin), and diarrheic shellfish poisoning (due to okadaic acid). Using traditional techniques of hapten (pure toxin) conjugation to protein to create complete antigen, animal immunization and antibody isolation, and specific antibody subpopulation purification, discriminating antibodies have been isolated that detect brevetoxins and ciguatoxin, but not okadaic acid, in a dose-dependent fashion. Using microorganic chemistry and purified toxins, a unique set of tools has been created for the study of polyether ladder toxin accumulation; depuration; and specific site localization in tissues, food sources, and clinical samples. Developed test protocols can detect toxin in dinoflagellate cells, in extracts from food sources, in seawater and culture media, and in human serum samples. Enzyme-linked immunosorbent assay protocols developed for eventual collaborative testing have been successful in limited applications within the laboratory (correlation coefficient of 0.92 excluding 2 outliers), and alternative formats are being developed to optimize the basic test for use in research laboratories, regulatory laboratories, and field inspections.

A new member of the gymnodimine class of spiroimine toxins has been isolated from a laboratory culture strain of Alexandrium ostenfeldii. Extensive one-dimensional (1D) and two-dimensional (2D) NMR data analysis was used to elucidate its structure as 12-methylgymnodimine B.

Strains of raphidophytes from different regions of the Gulf of California were established for identification and biochemical description. Raphidophyte in our coasts have been traditionally identified based on microscopic observations, a biochemical analyses of strains present in our coast is needed for a more detailed characterization and species confirmation. Strains were identified by morphological observations and sequencing of the 5.8S and COI regions. Cells were cultivated in modified f/2 media at 20°C with a light–dark cycle (12 h:12 h) and 150 μmol m−2 s−1 light intensity. Pigments were identified by HPLC, brevetoxins by LC–MS/MS, fatty acids by gas chromatography, superoxide radicals by spectrophotometry, and lipid peroxidation by the determination of thiobarbituric acid reactive substances. Strains were identified as Chattonella subsalsa, C. marina, and Fibrocapsa japonica. In all strains, the main pigment was chlorophyll a, followed by fucoxanthin, chlorophyll c1 and c2, violaxanthin, β-carotene, and diadinoxanthin. Strains were tested for PbTx-1, PbTx-2, PbTx-3, PbTx-6, PbTx-9, PbTx-carboxylic acid, brevenal, and brevisin; none were detected. All strains presented superoxide radical production and lipid peroxidation. The main fatty acids were 18:4 (n-3) and 20:5 (n-3). Strains had typical fatty acid composition for raphidophytes and produced brevetoxin-like compounds, had superoxide radical production, and lipid peroxidation. With this contribution, we confirm the presence of C. subsalsa, C. marina and F. japonica in the Gulf of California.

Blooms of the toxic alga Karenia brevis occur along coastlines where sessile suspension feeding invertebrates are common components of benthic communities. We studied the effects of K. brevis on four benthic suspension feeding invertebrates common to the coast of the SE United States: the sponge Haliclona tubifera, the bryozoan Bugula neritina, the bivalve Mercenaria mercenaria, and the tunicate Styela plicata. In controlled laboratory experiments, we determined the rate at which K. brevis was cleared from the seawater by these invertebrates, the effect of K. brevis on clearance rates of a non-toxic phytoplankton species, Rhodomonas sp., and the extent to which brevetoxins bioaccumulated in tissues of invertebrates using an enzyme-linked immunosorbent assay (ELISA). All four invertebrate species cleared significant quantities of K. brevis from seawater, with mean clearance rates ranging from 2.27 to 6.71 L g h⁻¹ for H. tubifera and S. plicata, respectively. In the presence of K. brevis, clearance rates of Rhodomonas sp. by B. neritina and S. plicata were depressed by 75% and 69%, respectively, while clearance rates by H. tubifera and M. mercenaria were unaffected. Negative effects of K. brevis were impermanent; after a recovery period of 13 h, B. neritina and S. plicata regained normal clearance rates. All four invertebrates accumulated high concentrations of brevetoxin after a 4h exposure to K. brevis, but when animals were transferred to filtered seawater for 15 h after exposure, brevetoxin concentrations in the tissues of H. tubifera and B. neritina decreased by ∼80%, while there was no change in toxin concentration in the tissues of S. plicata and M. mercenaria. High cell concentrations of K. brevis may cause a suppression of clearance rates in benthic suspension feeding invertebrates, resulting in a positive feedback for bloom formation. Also, high concentrations of toxin may accumulate in the tissues of benthic suspension feeding invertebrates that may be transferred to higher-level consumers.

The new pigment “moraxanthin” was found in natural samples from a fish mortality site in the Inland Bays of Delaware, USA. Pure cultures of the species, tentatively named Chattonella cf. verruculosa, and natural samples contained this pigment as a dominant carotenoid. The pigment, obtained from a 10 L culture of C. cf. verruculosa, was isolated and harvested by HPLC and its structure determined from MS and 1D- and 2D-NMR. The data identified this pigment as a new acylated form of vaucheriaxanthin called moraxanthin after the berry like algal cell. Its presence in pure cultures and in natural bloom samples indicates that moraxanthin is specific to C. cf. verruculosa and can be used as a marker of its presence when HPLC is used to analyze natural blooms samples.

Amphidinol 17 (AM17; 1), a novel amphidinol, has been isolated from a Bahamas strain of Amphidinium carterae. This new congener contains the signature hairpin region and a Delta(6) polyene arm, whereas the polyol arm is distinct from those of other amphidinols. The pattern of acetate incorporation in 1 was directly determined by feeding a single labeled substrate, [2-(13)C]acetate. While the highly conserved regions within the amphidinol family of AM17 have exhibited identical occurrences of cleaved acetates to other amphidinols for which the biosynthesis has been explored, the polyol arm for AM17 displays a higher degree of nascent chain processing that shows similarities to amphidinolide biosynthesis. AM17 exhibited an EC(50) of 4.9 microM in a hemolytic assay using human red blood cells but displayed no detectable antifungal activity.

This chapter provides a guide to how naked flagellates and coccolithophorids had their origin in the teachings and the “handout” literature developed for the advanced international phytoplankton course. It manifests a good image for the urgent need of such literature and portrays how the photocopies of the handouts began to appear in various laboratories around the world. It outlines how the literature was adapted, improved, and tested on the talented participants for each course, and offers an overview of how this goal was realized with an agreement to write one book containing this literature. There are several steps of the procedures that lead to the publication of this volume. It reviews how the idea of an international course in phytoplankton had its origin with Professor Trygve Braarud at the University of Oslo, Norway. The working group of phytoplankton methods was established during the executive meeting of the Scientific Committee in Oceanic Research (SCOR). The way the preparation of a phytoplankton manual of methodology started with the meeting at the University of Oslo under the auspice of SCOR in 1974 is also discussed.

The effect of light intensity and temperature on photosynthesis, chlorophyll a, and cellular concentrations of nitrogen and carbon in an axenic clone of Olisthodiscus luteus Carter following incubation in both indoor and outdoor growth chambers was determined. Populations were grown outdoors and photosynthesis determined in a graded series of light intensities yielding 4–524 total ly (per 6 h) and temperatures from 5 to 23 C and indoors at 0.3–40 total ly and 5–30 C. In both series, photosynthesis was saturated at low light (∼10 total ly or 0.028 ly · min −1) and showed only slight inhibition at the higher light intensities. Chlorophyll a·cell−1 had a maximum at 15 C with declining values at extremes of temperature. Carbon and nitrogen were more variable between indoor and outdoor experiments. Populations incubated indoors increased their carbon (130–225 pg·cell−1) and nitrogen (23–35 pg·cell−1) content as a function of light and temperature from 5 to 20 C. The outdoor series had increases in cellular N and C as a function of light and temperature below 14 C but maximum C·cell−1 (175 pg) and N·cell−1 (>27 pg) as well as wide variations in assimilation number occurred between 14–21 C. Assimilation numbers (

For 108 years marine botany research has been an important component of the research conducted at the Stazione Zoologica of Naples. The first researchers at the Naples Institute were German guests who proceeded with descriptive ecological and taxonomic studies and provided a foundation for the later physiological, cytological, life cycle, and biochemical studies conducted at the Stazione. During a major period (47 yrs.) Prof. G. Funk contributed ecological work giving the botanical research at Naples a continuity which extended into the late SO's. From 1960 onwards, the marine botany laboratory assumed a different direction and recently has returned to a physiological-ecological orientation. The lasting impact of marine botany has been the contribution of an ecological dimension from which other studies grew and found support. DIsCussIoN Studies of marine botany in the Gulf of Naples predate the founding of the Stazione Zoologica by nearly fifty years. The studies of Delle Chiaje (1823) and Costa (1838) describe species of the rich flora found in the waters of the kingdom of Naples. This flora and the equally abundant and varied fauna were factors influencing Anton Dohrn in establishing the Naples Institute. Within four years of the opening ofthe Stazione Zoologica, marine botany research was begun in earnest. For the past 108 years, research in this discipline has continued to contribute to the overall scientific effort. Marine botany and ecology are natural synonyms for the Stazione Zoologica, since from the beginning the ecological approach to the study of marine algae predominated, resulting in quantification and evaluation of the evolution of Neapolitan coastal waters. In addition, algal studies relating to cytology, physiology, anatomy, and aspects of biochemistry were pursued. Both micro and macro algae were studied although the major emphasis was placed on macroscopic thallate forms. The activity of marine botany research was greatly influenced by the perturbations imposed by the two world wars as well as the natural rhythms of the Institution's growth. The earliest visitors (1873—1900) were almost exclusively German researchers encouraged by Anton Dohrn to visit and work at his station. Among these, J. Reinke, P. Falkenberg, G. Berthold, R. Valiante, and C. Sauvageau were the first to extensively study the benthic algae of the Gulf. Armed with the modern elements of taxonomy and physiology, these early workers (Reinke, 1878a, b; Falkenberg, 1879, 1901; Berthold, 1882a, b; Valiante, 1883; Sauvageau, 1892) established vital species lists as well as distribution in the Naples area. In addition, their observations on gametes of brown algae, cellular composition including ions, chromoplasts, vacuoles, and associated membranes further added to the general knowledge of algae. The first decades of the 20th century had macroalgologists including A. Vickers, F. Tobler, E. Leick, and G. Funk as further contributors to our understanding of algal species distribution as related to ecological factors. Among these, the most

The distribution and abundance of the flagellate Olisthodiscus luteus Carter were examined over a 17 yr period (1959–75) at one station in lower Narragansett Bay, Rhode Island, and at many stations throughout the Bay during the 1974, 1975 summer bloom periods. A bimodal pattern of maximum abundance characterized the 17 yr data set. Maxima (103–104 cells·ml−1) occurred during June–July and October–November separated by a mid summer (August) minimum of 105 cells·ml−1 during June 1974 and were confined to the northern and western regions of Narragansett Bay. Incident light, temperature, salinity and nutrient (NH4+, NO3−, NO2−, PO43-) levels were also determined. The abundance cycle appears to be strongly effected by temperature, life cycle, motility, excreted substances and, to lesser extent, by light intensity, salinity, and nutrient dynamics. Regional distribution is affected by motility, wind forced water movements and benthic stage formation. The autecological data for O. luteus, pertinent observations of herbivorous zooplankton and the field observations are used to assess causative factors regulating the dynamics of O. luteus populations in Narragansett Bay. Pratt's hypothesis on the interaction between this species and Skeletonema costatum (Grev.) Cleve is reexamined.

Uptake and assimilation of nitrogen and phosphorus were studied in Olisthodiscus luteus Carter. A diel periodicity in nitrate reductase activity was observed in log and stationary phase cultures; there was a 10-fold difference in magnitude between maximum and minimum rates, but other cellular features such as chlorophyll a, carbon, nitrogen, C:N ratio (atoms) · cell−1 were less variable. Ks values (~2 μM) for uptake of nitrate-N and ammonium-N were observed. Phosphorus assimilated · cell−1· day−1 varied with declining external phosphorus concentrations; growth rates

The effect of salinity and temperature on Olisthodiscus luteus Carter has been examined to across the relative importance of these factory on dynamics of natural population. A salinity range 2–50% was observed with increased tolerance to low salinity ( 0.5 division day−1 at temperature of 15–30°C higher salinities (5–50%.) variable but distinct optima for density, growth and motility were observed 5, 10 and 30°C. Density and motility showed no clear optima from 10–10%.15–25°C where maximum growth rates >1.0 division/day−1 were common. Temperature increased from (0.5–1.9 division. Day−1) and increases of three orders of magnitude (102−103) for maximum densities. Temperature optima 20°C for growth 5–35%. And 25°C for >40%. were observed. The implications of these findings to natural populations of O. luleus are discussed.

The grazing of Acartia hudsonica and Acartia tonsa was observed on cultures of Olisthodiscus luteus and Dunaliella tertiotecta presented alone and in combination. Feeding on D. lertiolecla by both Acartia species increased directly with concentration but was always lower at equivalent food concentrations when O. luteus was present. In mixed phytoplankton cultures, A. hudsonica fed on both species at low O. luteus concentration, only on D, lertiolecla at intermediate levels and stopped feeding at highest O. luteus concentrations. A. tonsa showed a variable but similar result. These studies indicate the possible role of these dominant herbivores on the dynamics of O. luteus populations.